Cryptococcus neoformans is the etiological agent of human cryptococcosis. Cryptococcosis, as described in one textbook (Rippon, J. W., Medical Mycology, Saunders Co., Philadelphia, 1988), is a chronic, subacute or acute pulmonary, systemic, or meningitic yeast infection, generally beginning with a pulmonary infection. Its most serious clinical manifestation is in the central nervous system where it is the most prevalent agent of fungemic meningitis. Prior to the advent of amphotericin-B drug therapy, it was almost always fatal.
Cryptococcus neoformans, also known as Filobasidiella neoformans, is considered an opportunistic pathogen. The immune-compromised population, including AIDS/HIV infected individuals and cancer patients, is particularly susceptible to cryptococcosis. The organism is widely distributed in nature. The common pigeon, Columba livia, is a reservoir for cryptococcus. Cryptococcus neoformans can be recovered in large numbers from accumulated pigeon droppings in the birds' roosts.
Cryptococcus (also synonymous with the genus Filobasidiella) is a genus of Basidiomycetous yeasts. As such, herein they rill be referred to as "yeasts", "fungi", or "cryptococci". (Candida yeasts--the most clinically prevalent genus--are Ascomycetous yeasts.) There are several species of Cryptococcus; only C. neoformans is recognized as pathogenic. Cryptococcus albidus and C. laurentii are isolated from human clinical samples, but are not considered the causative agents of morbidity. Within C. neoformans, there are four known serotypes; A, B, C, and D. Serotype A is the most common pathogenic biotype.
Current diagnostic assays employ either India ink staining of clinical samples for microscope evaluation, or latex agglutination assays. Both technologies require high numbers of yeast cells in the sample.
It is an aspect of the present invention to provide nucleic acid probes which are specific for yeasts capable of causing cryptococcosis and related morbidity, particularly specific for the detection of Cryptococcus neoformans.
It is another aspect to provide probes which may be used in a variety of assay systems which avoid many of the disadvantages of the currently used detection methods.
It is still another aspect of the present invention to provide probes which can hybridize to target regions which can be rendered accessible to probes under normal assay conditions.
While Kohne at el. (Biophysical Journal 8:1104-1118, 1968) discuss one method for preparing probes to rRNA sequences, they do not provide the teaching necessary to sake Cryptococcus neoformans specific probes or any other probes to detect fungi.
Pace and Campbell (Journal of Bacteriology 107:543-547, 1971) discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin, Sogin and Woese (Journal of Molecular Evolution 1:173-184, 1972) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships. Fox, Pechman and Woese (International Journal of Systematic Bacteriology 27:44-57, 1977) discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. These references, however, fail to relieve the deficiency of Kohne's teaching with respect to fungi, and in particular, do not provide specific probes useful in assays for detecting cryptococcosis or its etiological agent, Cryptococcus neoformans.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Bacterial ribosomes contain three distinct RNA molecules which, at least in Escherichia coli, are referred to as 5S, 16S and 23S rRNAs. In eukaryotic organisms, there are four distinct rRNA species, generally referred to as 5S, 18S, 28S, and 58S. These names historically are related to the size of the RNA molecules, as determined by their sedimentation rate. In actuality, however, ribosomal RNA molecules vary substantially in size between organisms. Nonetheless, 5S, 18S, 28S, and 5.8S rRNA are commonly used as generic names for the homologous RNA molecules in any eukaryote, and this convention will be continued herein.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that alloy them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially, see next paragraph) to target nucleic acid sequences. In addition to their hybridization properties, probes also may contain certain constituents that pertain to their proper or optimal functioning under particular assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g. by end capping), to carry detection ligands (e.g. fluorescien, 32-P, biotin, etc.), or to facilitate their capture onto a solid support (e.g., polydeoxyadenosine "tails"). Such modifications are elaborations on the basic probe function which is its ability to usefully discriminate between target and non-target organisms in a hybridization assay.
Hybridization traditionally is understood as the process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion (one oriented 5' to 3', the other 3' to 5') to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another. The high specificity of probes relies on the low statistical probability of unique sequences occurring at random as dictated by the multiplicative product of their individual probabilities. These concepts are yell understood by those skilled in the art.
The stringency of a particular set of hybridization conditions is determined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids.
Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.
As a general matter, dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree.-65.degree. C. in a salt solution of approximately 0.9 molar. All references herein are fully incorporated by reference.